1
Mycochemical study of polysaccharides from edible mushroom Cortinarius caperatus (Gypsy mushroom) Zacchigna M. a , Altieri T. a , Beltrame G. a , Cateni F. a , Procida G. a a Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.zle Europa 1, 34127 Trieste, Italy E-mail: [email protected] Among basidiomycete molecules, cell wall polysaccharides have been recognized as a major class of bioactive constituents. [1] They are safe molecules and they have a wide spectrum of biological activities, such as immunostimulatory and antioxidant, therefore they possess a prominent role in health benefits coming from mushroom consumption. These properties make mushroom polysaccharides potential candidates for nutraceutical applications and bioactive ingredients production. [2] [1] - Zhang M. et al. (2007), Trends in Food Science & Technology 18, 4-19 [2] - Zhu F. et al (2015), Journal of Food Composition and Analysis 41, 165-173 PURIFICATION PROCEDURES Soluble Polysaccharide Structural elucidation of isolated polysaccharides has been performed using mono- and bi-dimensional NMR spectroscopy ( 1 H-NMR, 13 C-NMR, DEPT, 1 H- 1 H COSY, DQCOSY, TOCSY, HSQC, and HMBC), together with GC analysis of sylilated monosaccharides and methylation analysis [4] to determine the monosaccharide composition and the linkage type, respectively. [4] - Ciucanu I. & Kerek F. (1984), Carbohydrate Research, 131(2): 209- 217 INTRODUCTION STRUCTURAL ELUCIDATION OF ISOLATED POLYSACCHARIDES Insoluble Polysaccharide Fractionation of the hot aqueous extract of Cortinarius caperatus led to isolation of two polysaccharidic fractions characterized by spectroscopic analyses ( 1 H-NMR, 13 C- NMR, DEPT, 1 H- 1 H COSY, DQCOSY, TOCSY, HSQC, HMBC and HMQC), mass spectrometry (EI-MS, ESI-MS), infrared spectroscopy (FT-IR), chemical reactions of hydrolysis and derivatization followed by GC and HPLC analyses. [5] The mycochemical study revealed a water- soluble fraction characterized as β-(16)- D-glucan, whose presence inside C. caperatus has never, to the best of our knowledge, been reported before. Moreover, a purified water insoluble fraction has been characterized as a branched , glucan (Figures 3-5). [5] - Ruthes A. C. et al. (2015), Carbohydrate Polymers 117, 753-761 ANTIOXIDANT ACTIVITY The antioxidant activity of the soluble polysaccharide fraction has been evaluated as radical-scavenging activity by DPPH assay and the β-(16)-D-glucan showed significative antioxidant activity. RESULTS LIPIDIC FRACTION POLYSACCHARIDIC FRACTION [3] Dry methanolic extract Fraction D-DM CH 2 Cl 2 /MeOH 5:1 v/v Flash Chromatography Complex mixture of Triacylglycerlos TGAs Fraction D-DM 3-5 Ergosterol Complex mixture of Monoacylglycerols MAGs CH 2 Cl 2 CH 2 Cl 2 /MeOH 10:1 v/v Cortinarius caperatus Dry and milled mushroom Cold aq. extract Residue Residue Hot aq. extract H 2 O at 25°C, 6 h (x 3; 1,000 mL) H 2 O at 100°C , 6 h (x 3; 1,000 mL) Supernatant Precipitate EtOH precipitation (3:1, v/v) Soluble polysaccharide Freeze-thawing process Soluble fraction Insoluble fraction DMSO Treatment, at 60°C, 2 h Dialysis Freeze-thawing process Residue Branched α,β glucan [3] Smiderle F.R. et al. (2013), Carbohydrate Polymers, 94(1):9199 Fig. 1 1 H-NMR spectrum of the soluble polysaccharide β 1,6 glucan Fig. 2 HMBC spectrum of the soluble polysaccharide β 1,6 glucan Fig. 3 1 H-NMR of the branched α,β glucan Fig. 4 HSQC of the branched α,β glucan Fig. 5 TOCSY of the branched α,β glucan 0 20 40 60 80 100 0 500 1000 1500 2000 DPPH scavenging effect % microg/ml Soluble polysaccharide Ascorbic acid Dry extract Sugar linkage Residue C1 C2 C3 C4 C5 C6 6)-β-(1- A 103.40 73.64 76.06 70.01 75.40 69.33 6)-α-(1- A’ 100.70 73.55 -- 70.10 -- 68.76 α-(1- B 98.37 -- -- 69.11 -- 61.25 4,6)-β-(1- C 103.40 -- -- 76.14 -- 67.03

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Page 1: Mycochemical study of polysaccharides from edible mushroom ... · EtOH precipitation (3:1, v/v) Soluble polysaccharide Freeze-thawing process Soluble fraction Insoluble fraction DMSO

Mycochemical study of polysaccharides from edible

mushroom Cortinarius caperatus (Gypsy mushroom) Zacchigna M.a, Altieri T.a, Beltrame G.a, Cateni F.a, Procida G.a

aDepartment of Chemical and Pharmaceutical Sciences, University of Trieste, P.zle Europa 1, 34127 Trieste, Italy

E-mail: [email protected]

Among basidiomycete molecules, cell wall

polysaccharides have been recognized as

a major class of bioactive constituents. [1]

They are safe molecules and they have a

wide spectrum of biological activities, such

as immunostimulatory and antioxidant,

therefore they possess a prominent role in

health benefits coming from mushroom

consumption. These properties make

mushroom polysaccharides potential

candidates for nutraceutical applications

and bioactive ingredients production. [2]

[1] - Zhang M. et al. (2007), Trends in Food Science & Technology 18,

4-19

[2] - Zhu F. et al (2015), Journal of Food Composition and Analysis 41,

165-173

PURIFICATION PROCEDURES Soluble Polysaccharide

Structural elucidation of isolated

polysaccharides has been performed using

mono- and bi-dimensional NMR

spectroscopy (1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, DQCOSY, TOCSY, HSQC,

and HMBC), together with GC analysis of

sylilated monosaccharides and methylation

analysis [4] to determine the

monosaccharide composition and the

linkage type, respectively. [4] - Ciucanu I. & Kerek F. (1984), Carbohydrate Research, 131(2): 209-

217

INTRODUCTION STRUCTURAL ELUCIDATION

OF ISOLATED

POLYSACCHARIDES

Insoluble Polysaccharide Fractionation of the hot aqueous extract of

Cortinarius caperatus led to isolation of

two polysaccharidic fractions characterized

by spectroscopic analyses (1H-NMR, 13C-

NMR, DEPT, 1H-1H COSY, DQCOSY,

TOCSY, HSQC, HMBC and HMQC), mass

spectrometry (EI-MS, ESI-MS), infrared

spectroscopy (FT-IR), chemical reactions

of hydrolysis and derivatization followed by

GC and HPLC analyses. [5] The

mycochemical study revealed a water-

soluble fraction characterized as β-(16)-

D-glucan, whose presence inside C.

caperatus has never, to the best of our

knowledge, been reported before.

Moreover, a purified water insoluble

fraction has been characterized as a

branched 𝛼,𝛽 glucan (Figures 3-5).

[5] - Ruthes A. C. et al. (2015), Carbohydrate Polymers 117, 753-761

ANTIOXIDANT ACTIVITY

The antioxidant activity of the soluble

polysaccharide fraction has been evaluated

as radical-scavenging activity by DPPH

assay and the β-(16)-D-glucan showed

significative antioxidant activity.

RESULTS

LIPIDIC FRACTION

POLYSACCHARIDIC FRACTION[3]

Dry

methanolic

extract

Fraction D-DM

CH2Cl2/MeOH 5:1 v/v

Flash

Chromatography

Complex

mixture of

Triacylglycerlos

TGAs

Fraction D-DM

3-5

Ergosterol

Complex mixture of

Monoacylglycerols MAGs

CH2Cl2 CH2Cl2/MeOH 10:1 v/v

Cortinarius caperatus

Dry and milled mushroom

Cold aq. extract Residue

Residue Hot aq. extract

H2O at 25°C, 6 h

(x 3; 1,000 mL)

H2O at 100°C , 6 h

(x 3; 1,000 mL)

Supernatant Precipitate

EtOH precipitation (3:1, v/v)

Soluble

polysaccharide

Freeze-thawing process

Soluble

fraction

Insoluble fraction

DMSO Treatment, at 60°C, 2 h

Dialysis

Freeze-thawing process

Residue Branched α,β glucan

[3] Smiderle F.R. et al. (2013), Carbohydrate Polymers, 94(1):91– 99

Fig. 1 1H-NMR spectrum of the soluble polysaccharide β

1,6 glucan

Fig. 2 HMBC spectrum of the soluble polysaccharide β 1,6

glucan

Fig. 3 1H-NMR of the branched α,β glucan

Fig. 4 HSQC of the branched α,β glucan

Fig. 5 TOCSY of the branched α,β glucan

0

20

40

60

80

100

0 500 1000 1500 2000 DP

PH

scavengin

g e

ffect

%

microg/ml

Soluble polysaccharide

Ascorbic acid

Dry extract

Sugar linkage

Residue C1 C2 C3 C4 C5 C6

6)-β-(1- A 103.40 73.64 76.06 70.01 75.40 69.33

6)-α-(1- A’ 100.70 73.55 -- 70.10 -- 68.76

α-(1- B 98.37 -- -- 69.11 -- 61.25

4,6)-β-(1- C 103.40 -- -- 76.14 -- 67.03